The present invention relates to pharmaceutical preparations which comprise a botulinum neurotoxin from Clostridium botulinum, the neurotoxin being free of the complexing proteins naturally present in the complex. The direct consequence thereof is the realization, on which the present invention is based, that with the free neurotoxin, in contrast to the complex, there is only a distinctly reduced, or no, induction of neutralizing antibodies in the patient. The present invention further relates to the use of botulinum neurotoxins from Clostridium botulinum for producing a medicine for treating disorders of the nervous system. Another aspect of the present invention relates to the use of the botulinum neurotoxins from Clostridium botulinum for cosmetic treatment.
Clostridium botulinum toxin complex type A (Mr 900,000) has been employed for several years for the therapy of various dystonias. At present two different products comprising this complex are approved for the treatment of blepharospasm, hemifacial spasms and spasmodic torticollis: BOTOX® and DYSPORT®. Clinical trials of the therapy of other disorders of the nervous system (e.g. spasticities, migraine, low back pain, cervical spine disorders, hypersalivation) are currently in progress. The products are also employed for cosmetic indications such as hyperhidrosis and pronounced wrinkling. The other Clostridium botulinum toxin complexes (of types B, C, D, E, F, G) are also suitable for these therapies. However, at present there is no approved product comprising one of the type B-G toxins on the market.
Botulinum toxin complexes are composed of a mixture of clostridial proteins. These are hemagglutinins with different molecular masses, a nontoxic, non-hemagglutinating protein (Mr about 120,000) and a neurotoxin (Mr about 150,000). They form an acid-stable complex which is responsible for the oral toxicity in cases of food poisoning. In contrast to the pure neurotoxin, the complex resists the aggressive environment in the gastrointestinal tract and makes enteral absorption of the neurotoxin possible, and this reaches the target cells via the bloodstream or the lymphatic system and there induces blockade of transmitter release. This is followed by a paralysis of striped and smooth muscles and cessation of various autonomic functions. Poisoned patients die of respiratory muscle failure. Since the pure neurotoxin is degraded in the gastrointestinal tract and thus does not undergo enteral absorption, it is not toxic after ingestion. On parenteral administration, the therapeutic effects of the neurotoxin and of the complex do not differ since the complex decomposes into its constituents in tissue, and only the neurotoxin is taken up by the target cells.
For therapeutic use, the complex is in the current state of the art injected directly into dystonic or spastic muscles, where the neurotoxin is released at physiological pH from the complex and elicits the desired pharmacological effect. Although the complex is administered only in extremely low doses (1-25 ng, depending on indication and size of the affected muscle), repeated injection is followed in a considerable number of patients by formation of specific neutralizing antibodies which are also directed against the neurotoxin. The direct consequence is that antibody-positive patients no longer respond to the complex. However, they might be treated with other toxin types, although none of them is approved for therapy. When the patient has been tested with all the toxin types and has formed antibodies against them, further administration of a botulinum toxin complex (irrespective of the type) no longer provides a remedy. It must be taken into account in this connection that each dose of complex contributes to increasing the antibody titer until further administration of the complex no longer makes sense because no effect is now achieved. It often takes years for the antibody titer to fall significantly, so that these patients are not (cannot be) treated (with botulinum neurotoxin) for long periods.
The formation of specific antibodies is favored by two factors. On the one hand, the neurotoxin, fixed in the complex, remains in the tissue for a long period and may activate immune cells which migrate into the tissue to form antibodies. The long residence time does not result in increased uptake by the target cells, however, since poisoned target cells are no longer able to take up toxin. The neurotoxin which slowly dissociates out of the complex thus now has only immunological activity. On the other hand, the proteins present in the complex intensify an immune response. Hemagglutinins are lectins, that is to say proteins which are distinguished by a high affinity for certain sugars. Because of their binding to sugar structures, lectins have immunostimulating effects. Thus, it has been possible to show that the lectins concanavalin A, phytohemagglutinin and pokeweed mitogen activate T and B lymphocytes. The hemagglutinins of the botulinum toxin complexes, which likewise bind to membrane-associated sugars, are thus able in a similar way to act as immunoadjuvants and contribute to antibody formation and thus to failure of the therapy.